19th February 2024

Basics of Genetics and Gene Analysis

Basics of genetics and its abnormalities

Basics of genetics

DNA

The nucleus of the human cells contains 46 chromosomes, two pairs of autosomal chromosomes with two sex chromosomes, known as diploid. Human chromosomes consist of deoxyribonucleic acid (DNA) wound in a double helix. Ribonucleic acid (RNA) is a single-stranded molecule that mediates protein formation from DNA. Three main subtypes exist:

• Messenger RNA (mRNA) – Formed during transcription and forms the template for protein synthesis.
• Transfer RNA (tRNA) – Transport molecules that interact with the mRNA within the ribosome. An amino acid is attached to one end of the tRNA molecule. During translation, tRNA adds the amino acid to the polypeptide chain.
• Ribosomal RNA (rRNA) – Component of the ribosome organelle. Aids in the binding of mRNA to tRNA during translation.

There are several types of DNA. Single-copy DNA accounts for half of the human genome and accounts for the introns and exons. The remainder of the human genome consists of repetitive DNA. Repetitive DNA can be divided into:

• Dispersed repetitive DNA – Found as short, interspersed elements and long interspersed elements. They function as polymorphisms and are randomly distributed within the genome.
• Satellite DNA – Clustered repetitive segments of tandem repeats with varying lengths (tens to thousands of base pairs). Useful for genetic mapping due to variable sizes between individuals.

Mitochondrial DNA is a circular genome that encodes proteins in the mitochondrial respiratory chain and specific transfer RNAs. Mitochondrial DNAs are derived from the ovum as no mitochondria are transferred from the spermatozoa during fertilisation.

Cell cycle

Normal cell division occurs by mitosis, resulting in 2 genetically identical daughter cells.

Mitosis occurs in 4 distinct phases: prophase, metaphase, anaphase, and telophase.

Image Source: Wikimedia Commons (2022). Mitosis schematic diagram, Licensed under a creative commons licence. Available for free at: https://commons.wikimedia.org/wiki/File:Mitosis_schematic_diagram-en.svg (Accessed: 4 January 2024).

Reproductive cells divide by meiosis, resulting in daughter cells containing one chromosome of each chromosome pair, known as the haploid daughter cells. Genetic diversity occurs through a process of “crossing-over”, where segments of homologous chromosomes are exchanged. Meiosis occurs in 2 distinct steps, meiosis I and II. Homologous chromosomes are divided during meiosis I, forming the haploid cells, while sister chromatids are separated during meiosis II.

Image Source: Wikimedia Commons (2016). Meiosis Stage, Licensed under a creative commons licence. Available for free at: https://commons.wikimedia.org/wiki/File:Meiosis_Stages.svg (Accessed: 4 January 2024).

Further details can be obtained from the cell cycle section.

Genetic abnormalities

Chromosomal abnormalities or base-pair mutations can cause genetic mutations. Chromosomal abnormalities can be broadly divided into numerical abnormalities and structural abnormalities.

Numerical abnormalities are caused by the failure of chromosomal separation during meiosis and can manifest as:

• Error in ploidy – Failure of the division of the entire set of homologous chromosomes during anaphase I or failure of the division of all sister chromatids during anaphase II, resulting in a diploid gamete. Fertilisation with a normal gamete results in a triploid cell and is usually incompatible with life.
• Aneuploidy – Occurs due to a failure to separate a single chromosome pair during anaphase, resulting in monosomic or trisomic cells following fertilisation.

Structural abnormalities occur during prophase I as chromosomes undergo crossing over. This can manifest as:

• Reciprocal translocation – 2 non-homologous chromosomes swapped segments. This can result in trisomy or monosomy of the translocated segments.
• Robertsonian translocation – Translocation affects acrocentric chromosomes (chromosomes with centromeres close to the end of the chromosome), forming fusion chromosomes. Carriers of such translocations can be asymptomatic due to the balanced nature of the translocation. Increased risk of monosomy or trisomy for their offspring.
• Deletion – Segments of chromosomes are lost during crossing-over, which usually results in significant abnormalities.
• Insertion – Chromosome segments are added during crossing-over, usually resulting in less significant abnormalities.
• Inversion – Error in chromosomal rearrangement where a segment within the chromosome is reversed.

Base-pair mutations occur during DNA replication errors or by environmental factors, resulting in a change in the DNA sequence, and can result in single-gene disorders. This manifests in several ways:

• Base-pair substitution – Single substitution of a nucleotide with a different base-pair. This does not typically result in a phenotype change due to nonsense DNA and redundancies within the codons that encode amino acids. This can result in a missense mutation, resulting in the premature termination of a polypeptide sequence.
• Frame-shift mutation – Occurs following insertion or deletion of a nucleotide, resulting in alteration of the amino acid sequence downstream from the mutation.
• Trinucleotide expansion mutation – Characterised by amplification of a sequence of 3 nucleotides. Dynamic mutations can increase with gametogenesis, affecting the phenotypic presentation.

References

• Trent, R. J. (2012). Chapter 1 – Genes to Personalised Medicine. In R. J. Trent (Ed.), Molecular Medicine (Fourth Edition) (pp. 1–37). Boston/Waltham: Academic Press.
• Read, A., & Donnai, D. (2021). Chapter 2: How do genes work? In New Clinical Genetics: A guide to genomic medicine (4ᵗʰ ed.). United Kingdom: Scoin Publishing Limited.
• Strachan, T., & Read, A. P. (2019). Part 1: Basics of DNA, Chromosomes, Cells, Development, and Inheritance. In Human Molecular Genetics (5ᵗʰ ed.). United States: CRC Press.
• Jorde, L. B., Carey, J. C., & Bamshad, M. J. (2016). Chapter 1: Basic Cell Biology. In Medical Genetics (5ᵗʰ ed.). United States: Elsevier.
• Maitra, A., Kumar, V., Abbas, A. K., & Aster, J. C. (2013). Genetic and Paediatric Diseases. In Robbin’s Basic Pathology (9ᵗʰ ed.): Elsevier.
• Kelsell, D., Bryne, C., & Linton, K. (2012). Molecular cell biology and human genetics. In P. Kumar & M. Clark (Eds.), Kumar and Clark’s Clinical Medicine. Spain: Elsevier Saunders.

Methods of gene analysis

Background

DNA analysis is critical to aid with the diagnosis of genetic disorders. Recent developments in scientific methods have altered how genetic mutations are investigated, and this remains an evolving field. Candidates are not expected to know specific details but to understand the basic techniques of detecting genetic mutations.

Samples of a patient’s tissue are required for testing, and this can be obtained from blood, buccal mucosa or within affected tissues. Prenatal testing for foetal genetic abnormalities can be performed on amniotic fluid or chorionic villus samples. Genetic testing can be carried out in a variety of methods:

Karyotyping

Karyotyping involves whole chromosomal analysis using microscopy. Cells are cultured within a medium, and colchicine arrests cell division during metaphase. The chromosomes are made visible using various stains and arranged based on their appearance to form the karyotype.

Image Source: Wikimedia Commons (2017). Peripheral blood karyotype result belonging to the subject compatible with 48-XYYY, Licensed under a creative commons licence. Available for free at: https://commons.wikimedia.org/wiki/File:Peripheral-blood-karyotype-result-belonging-to-the-subject-compatible-with-48-XYYY.png (Accessed: 4 January 2024).

Karyotypes have a role in identifying large genetic abnormalities, including aneuploidy, reciprocal and Robertsonian translocation, and large microdeletions or duplications. Its use has largely been superseded by microarray analysis but still has a role in identifying balanced translocations.

Nucleic acid hybridisation

Hybridisation detects mutations using single-stranded DNA probes to detect specific DNA/RNA sequences within the genetic material.

Several methods utilise hybridisation to detect mutations:

• Southern blotting – DNA samples are broken down into smaller fragments, and electrophoresis is employed to separate DNA fragments based on size. DNA probes with a marker are applied to allow hybridisation, and chemiluminescence or X-rays are used to detect the probes. This only detects significant mutations or DNA sequences that PCR cannot amplify.
• Fluorescence in-situ hybridisation (FISH) – Single-stranded DNA probes are exposed to denatured chromosomes within the interphase, prophase or metaphase stage of cell division, and fluorescence is used to detect its bonding to sample DNA. FISH occasionally detect aneuploidies, translocations, and microdeletions/duplications.

Image Source: Wikimedia Commons (2009). Hordeum vulgare stained by fluorescent in situ hybridization, Licensed under a creative commons licence. Available for free at: https://commons.wikimedia.org/wiki/File:Hordeum_vulgare_stained_by_fluorescent_in_situ_hybridization.jpg  (Accessed: 4 January 2024).

• Array comparative genomic hybridisation (aCGH) – A microarray containing thousands to millions of oligonucleotide probes is exposed to a sample and a control DNA sample. Both DNA samples are marked with a fluorescent dye and are compared under a fluorescence microscope. This method detects aneuploidy, deletions, duplications (both large and small) and unbalanced translocations. It is possible to detect single nucleotide polymorphism with different probes. aCGH rarely detects balanced translocation as no excess genetic material exists within the sample DNA.

Image Source: Wikimedia Commons (2012). Array-CGH protocol, Licensed under a creative commons licence. Available for free at: https://commons.wikimedia.org/wiki/File:Array-CGH_protocol.svg (Accessed: 4 January 2024).

Polymerase chain reaction (PCR)

PCR is performed by denaturing the sample DNA and annealing a DNA primer to the denatured DNA to allow expression of the sequence of interest. DNA polymerase is used to amplify the sequence, exponentially increasing the amount of the DNA sequence of interest available. PCR is often only successful in sequences within the hundreds to thousands of base pairs, with sequences over 20kbp almost impossible to amplify.

Gene analysis uses various methods, including electrophoresis, restriction enzyme analysis, allele-specific oligonucleotide hybridisation and multiplex ligation-dependant probe amplification. PCR is versatile, detecting insertions, deletions, point mutations and trinucleotide expansion mutations.

DNA sequencing

DNA sequencing determines the precise sequence of A, C, T and G nucleotides within the gene of interest. It is helpful in detecting insertion, deletion, point mutations and nucleotide polymorphisms. At its core, sequencing is performed using either the Sanger method or next-generation sequencing (NGS).

The Sanger method analyses a DNA sequence by starting with multiple DNA strands and a primer to target a sequence of interest. Dideoxynucleoside molecules terminate the nucleotide chain during the reaction, creating multiple chains of DNA with differing lengths. Each dideoxynucleoside is attached to its fluorescent dye, and a computer program compares each fragment’s size and the fluorescent dye present to determine the precise DNA sequence. This process is labour-intensive but allows a specific gene to be sequenced accurately.

NGS allows rapid sequencing of an entire genome. The genome is divided into millions of fragments, each up to several hundred base pairs in size. Fragments are amplified using PCR, complementary base pairs are attached, and laser scanning helps to identify the fluorescent dye. It is useful to detect rare genetic diseases caused by mutations of various genes.

References

• Ratan, Z. A., Zaman, S. B., Mehta, V., Haidere, M. F., Runa, N. J., & Akter, N. (2017). Application of Fluorescence in Situ Hybridization (FISH) Technique for the Detection of Genetic Aberration in Medical Science. Cureus, 9(6), e1325. https://doi.org/10.7759/cureus.1325
• Wallace SE, Bean LJH. Educational Materials — Genetic Testing: Current Approaches. 2017 Mar 14 [Updated 2020 Jun 18]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK279899/
• Gomes, A., & Korf, B. (2018). Chapter 5 – Genetic Testing Techniques. In N. H. Robin & M. B. Farmer (Eds.), Pediatric Cancer Genetics (pp. 47-64): Elsevier.
• Turnpenny, P. D., & Ellard, S. (2017). Laboratory Techniques for Diagnosis of Monogenic Disorders. In Emery’s Elements of Medical Genetics. China: Elsevier.
• Kingston, H. M. (2002). Techniques of DNA analysis. In ABC of Clinical Genetics. Malaysia: BMJ Books.
• Jorde, L. B., Carey, J. C., & Bamshad, M. J. (2016). Genetic Variation: Its Origin and Detection. In Medical Genetics (5ᵗʰ ed.). Canada: Elsevier.
• Read, A., & Donnai, D. (2021). Chapter 4: How can a patient’s DNA be studied? In New Clinical Genetics: A guide to genomic medicine (4ᵗʰ ed.). United Kingdom: Scoin Publishing Limited.